Journal:

Article Title: LAT1 Expression in Pre and Post Implantation Embryos and Placenta

doi: 10.1016/j.placenta.2008.12.015

Figure Lengend Snippet: RT-PCR product encoding LAT1 was detected in cDNA from one (I) embryo equivalent at timed stages of development 1C, 1-cell; 2C, 2-cell; 4C, 4-cell; 8C, 8-cell; M, morula; B, blastocyst; HB, hatching blastocyst (A). Representative image of three independent replicates. Quantitative analysis of LAT1 are shown in (B). mRNA transcript levels in a developmental series of mouse preimplantation embryos by Real-Time RT-PCR. Data were normalized to external Luciferase control (0.5pg/embryo) and relative to 4-cell target gene mRNA levels. Relative mRNA levels are presented as the mean ± s.e, representative of three independent replicates. Bars with different letters represent significant differences in relative mRNA levels between embryo stages (p≤0.05).

Article Snippet: First-strand complementary DNA (cDNA) synthesis was performed using random hexamers and the Supe r Script TM First-Strand Synthesis System for RT-PCR (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions.

Techniques: Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Luciferase, Control

Journal: mBio

Article Title: Unraveling the cryptic functions of mitogen-activated protein kinases Cpk2 and Mpk2 in Cryptococcus neoformans

doi: 10.1128/mbio.01156-24

Figure Lengend Snippet: Mpk2 functions as an Mpk1 paralog for maintaining the CWI. ( A ) The wild-type strain (H99) was grown to an OD 600 of 0.8 before transitioning to a fresh yeast extract-peptone-dextrose (YPD) medium containing 1% CFW. Cells were harvested at intervals of 0, 30, 60, 90, and 120 min and subjected to RNA extraction and cDNA synthesis. Expression levels of MPK1 and MPK2 were determined via quantitative reverse transcription-PCR (qRT-PCR) in three biological replicates. ( B ) MPK2 expression levels were quantified by qRT-PCR with three biological replicates in the wild-type (H99) and mpk1 Δ P H3 :MPK2 (YSB9585 and YSB9586) strains. ( C ) Following overnight growth, cells from wild-type (H99), mpk1 Δ (KK3), mpk2 Δ (YSB3236), mpk1 Δ mpk2 Δ (YSB8245), and mpk1 Δ P H3 :MPK2 (YSB9575) strains were serially diluted to 10 −4 and spotted on solid media containing various stressors: 0.03% Congo Red (CR), 2.5 mg/mL CFW, 32 µg/mL anidulafungin, or 32 µg/mL micafungin. After 2 days, the plates were imaged. ( D ) Wild-type, mpk1 Δ, mpk2 Δ, and mpk1 Δ mpk2 Δ strains were cultured overnight, synchronized to OD 600 0.2, and further grown to 0.8 with fresh YPD. The strains were then treated with 1% CFW for 0, 30, and 60 min. After specified times, 40 mL of cells was collected and frozen with liquid nitrogen. Whole proteins were extracted and immunoblotted with phospho-p44/42 antibody. Phosphorylated Mpk1 and Mpk2 levels were measured using the chemiluminescence method, after which the membranes were stripped and reprobed with anti-actin antibody to quantify total proteins. Protein quantification was conducted using the ImageLab program. ( E ) Wild-type, mpk1 Δ, mpk2 Δ, mpk1 Δ mpk2 Δ, and mpk1 Δ P H3 :MPK2 strains were cultured overnight and adjusted to an OD 600 of 0.2 and then grown for 24 h in YPD at 30°C and fixed with formaldehyde solution. Cells were stained with CFW in the dark, washed, and imaged using differential interference contrast (DIC) and DAPI (4′,6-diamidino-2-phenylindole) filters. Chitin content in the membrane of randomly chosen 50 cells was assessed using ImageJ. ( F ) The aforementioned strains in panels D and E were grown in capsule-inducing Littman medium for 2 days. Cells were then stained with India ink, and the capsular thickness of 50 randomly selected cells was measured using NIS-elements software. The indicated white scale bar is 10 µm. Statistical differences were determined using one-way ANOVA and Tukey’s test: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; NS = not significant.

Article Snippet: First antibodies were used as follows: phospho-p44/42 MAPK antibody (Cell signaling, #4370, 2,000:1), anti β-actin antibody (sc-47778, 1,000:1), and anti α-tubulin antibody (sc-8035, 1,000:1).

Techniques: RNA Extraction, cDNA Synthesis, Expressing, Reverse Transcription, Quantitative RT-PCR, Cell Culture, Staining, Membrane, Software

Journal: mBio

Article Title: Unraveling the cryptic functions of mitogen-activated protein kinases Cpk2 and Mpk2 in Cryptococcus neoformans

doi: 10.1128/mbio.01156-24

Figure Lengend Snippet: Mpk2 is regulated by MAP2K Mkk2 and MAP3Ks Ssk2 and Ste11. ( A and B ) Wild-type, mpk1 Δ, mpk2 Δ, bck1 Δ (YSB273), mkk2 Δ (YSB330), ssk2 Δ (YSB264), and ste11 Δ (YSB313) strains were grown overnight, resuspended with fresh yeast extract-peptone-dextrose (YPD) medium at an OD 600 of 0.2, and further incubated to an OD of 0.8. Subsequently, these strains underwent a treatment with 1% CFW for intervals of 0, 30, and 60 min. Following each designated time point, 40 mL of the cell cultures was spun down and flash-frozen in liquid nitrogen. Total proteins were then extracted for immunoblot analysis using an anti phospho-p44/42 antibody. The levels of phosphorylated Mpk1 and Mpk2 were detected by chemiluminescence. After detection, the blots were stripped and re-probed for actin or tubulin to assess the total protein levels. ( C ) A schematic of the proposed Mpk1/Mpk2 regulatory mechanisms for maintaining CWI in C. neoformans .

Article Snippet: First antibodies were used as follows: phospho-p44/42 MAPK antibody (Cell signaling, #4370, 2,000:1), anti β-actin antibody (sc-47778, 1,000:1), and anti α-tubulin antibody (sc-8035, 1,000:1).

Techniques: Incubation, Western Blot

Journal: BMC Plant Biology

Article Title: Elicitor-induced transcription factors for metabolic reprogramming of secondary metabolism in Medicago truncatula

doi: 10.1186/1471-2229-8-132

Figure Lengend Snippet: Expression of WRKY transcription factors in M. truncatula cell cultures . A, induction of WRKYs by YE as revealed by oligonucleotide array analysis. The double apostrophes represent minutes and the single apostrophes represent hours. B, WRKY transcript levels in YE and MJ treated cells determined by Affymetrix array analysis. C, Detailed time course for WRKY gene transcript levels in response to YE, as determined by RT-PCR. Actin is shown as loading control.

Article Snippet: Three μg of total RNA was used in a first strand synthesis using Ready-To-Go RT-PCR Beads (Amersham Biosciences Corp, Pittsburgh, PA) in a 50 μl reaction with oligo-dT primers according to the manufacturer's protocol.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: BMC Plant Biology

Article Title: Elicitor-induced transcription factors for metabolic reprogramming of secondary metabolism in Medicago truncatula

doi: 10.1186/1471-2229-8-132

Figure Lengend Snippet: RT-PCR analysis of Medicago W109669 and tobacco PR2 transcript levels in transgenic tobacco lines overexpressing W109669 . Control plants harbored pBI121. Control and transgenic plants of the T 0 generation were used for analysis. Actin is shown as loading control.

Article Snippet: Three μg of total RNA was used in a first strand synthesis using Ready-To-Go RT-PCR Beads (Amersham Biosciences Corp, Pittsburgh, PA) in a 50 μl reaction with oligo-dT primers according to the manufacturer's protocol.

Techniques: Reverse Transcription Polymerase Chain Reaction, Transgenic Assay